This invention relates to shuttle vectors for inserting DNA in mycobacteria.
It also relates to the use as vaccines of these mycobacteria in which one of these vectors has been inserted.
The Mycobacterium genus comprises over 50 species, including major pathogens for the human such as Mycobacterium tuberculosis, agent of tuberculosis, Mycobacterium leprae, agent of leprosy, and Mycobacterium avium, a major opportunist in the course of AIDS. Little is known of the genetic mechanisms of the virulence of M. tuberculosis and the other pathogenic species for the human, due to the low growth rate of these bacteria and the lack of adequate cloning vectors; and as a result it is difficult to insert foreign DNA in these microorganisms. Moreover, the mycobacteria show a particular resistance to many antibiotics due to the fact that the constitution of their wall is poorly permeable to these molecules. However, recent studies have shown that it is possible to introduce foreign DNA in BCG with a view to expressing antigens heterologous to this bacterium.
In the case of BCG manipulation, the use of markers of resistance to antibiotics hitherto available in mycobacteria such as the aph3 gene providing a high degree of resistance to kanamycin or genes providing resistance to streptomycin or spectinomycin, cannot be envisaged because it could cause a dissemination of resistance to these antibiotics in the environment in the event that recombinant BCG is used for therapeutic or prophylactic purposes in the human or the animal. It is thus of the utmost importance to be able to develop resistance markers which are harmless for the environment in order to select recombinant mycobacteria which can be widely used.
At the present time, only a very small number of markers usable in mycobacteria is available.
Thus, Aldovini et al. (J. Bacteriol, 1993, 175, 7282-7289) have isolated the gene coding for orotidine-5xe2x80x2-monophosphate decarboxylase (OMP-DCase) of Mycobacterium bovis BCG.
Another marker, the cI gene, coding for the L1 phage repressor, has also been used (patent application WO-90/00594).
Shuttle vectors based on these markers and allowing the transfer of DNA between Escherichia coli and Mycobacterium have already been described. Thus, patent application WO-91/13 157 describes two shuttle vectors, pEP2 and pEP3, respectively carriers of a gene of resistance to kanamycin and hygromycin.
One of these vectors, pEP2, is modified by the introduction of the protein promoter MBP70 of Mycobacterium, as described in patent application WO-90/10 701.
Other shuttle vectors usable in mycobacteria and carrying markers of resistance to antibiotics, auxotrophic markers or the cI gene have been described in patent application WO 90/00594.
Finally, Ranes et al. (J. Bacteriol. 1990, 172, 2793-2797) have described the construction of a shuttle vector called pRR3 carrying a gene of resistance to kanamycin expressing itself in Mycobacterium bovis BCG and Mycobacterium smegmatis. 
A strain of non-pathogenic saprophytic mycobacterium resistant to mercury carrying a plasmid of 115 MD was also known (Meissner and Falkinham, J. Bacteriol, 157, 669-672, 1984). This very large plasmid was not, however, characterized. In particular, the genes involved in the resistance to mercury were neither localized nor identified, and consequently this plasmid could not be used as a shuttle plasmid. Moreover, saprophytic mycobacteria do not belong to pathogenic mycobacteria, the latter being principally included in the M. tuberculosis, M. bovis and M. leprae species. 
The result was that those skilled in the art wishing to transfer DNA coding for antigens, obtained by cloning in Escherichia coli, to mycobacteria with a view to fabricating vaccines, was confronted with a lack of easy-to-manipulate vectors carrying markers expressing themselves in E. coli and mycobacteria, and without danger to human or animal health. The use of genes of resistance to antibiotics of therapeutic aim must, in fact, be ruled out for the reasons given above. The use of auxotrophic mutants gives rise to practical difficulties of selection and moreover involves the need to obtain mutants for each mycobacterium strain.
The applicants therefore set out to find other markers which could be included in shuttle vectors without excessively reducing the ability of said vectors to include other DNA fragments, and which would provide on the mycobacteria in which they are expressed characters making it possible to distinguish them, in a way which is both unambiguous and easy to implement, from mycobacteria not carrying these markers.
The applicants have thus surprisingly revealed that shuttle vectors originating from Gram-negative bacteria, carrying genes of resistance to a compound containing a heavy metal such as inorganic mercury or. mercurial compounds, were expressed in Gram-positive bacteria and could be used for the transfer of DNA between Escherichia coli and Mycobacterium, and that bacteria carrying these shuttle vectors could be selected easily.
This invention therefore relates to a shuttle vector for inserting DNA in mycobacteria comprising at least one origin of functional replication in said mycobacteria, another origin of functional replication in other bacteria, such as E. coli, an enzyme cutting site allowing the insertion of DNA coding for a protein capable of being expressed in the mycobacteria, characterized in that it also carries a gene providing on said mycobacteria resistance to a compound containing a heavy metal such as inorganic mercury, a mercurial compound or arsenic.
It should be considered that those skilled in the art have for many years been confronted with the problem of seeking markers for the transfer of genes in mycobacteria, but that up to now no satisfactory solution has been found to this problem.
The solution found by the applicants, namely the choice of genes originating from Gram-negative bacteria and coding for a resistance to a heavy metal, is particularly surprising in view of the fact that those skilled in the art would not in theory have expected to find Gram-negative bacteria expressed in Gram-positive bacteria. Indeed, many studies have demonstrated the inability of genes from Gram-negative bacteria to express themselves in Gram-positive bacteria (Misra, Plasmid, 27, 4-16, 1992; Robinson and Tuovinen, Microbiological Reviews, Vol. 48 No. 2, 95-124, 1984).
The applicants have thus found a solution to a long-standing problem and one which, moreover, is of great importance to public health: mycobacteria and in particular M. bovis BCG, are considered as good candidates for vaccination against various diseases, provided that it is possible for these bacteria to express foreign antigens. This invention provides for an introduction of genes coding for these antigens which is both efficient and without danger to human health.
This invention therefore involves a major technical advance in the field of public health.
It should also be noticed that although markers of resistance to mercury were already known to exist, particularly in mycobacteria, those skilled in the art were in no way incited to integrate genes of resistance to mercury or mercurial compounds in shuttle vectors usable in mycobacteria. On the contrary, it is widely known that operons expressed for example in E. coli are not expressed in mycobacteria. Those skilled in the art were thus not incited to use in mycobacteria genes usually expressed in E. coli. 
In other words, nothing suggested using genes of resistance, in particular to mercury or mercurial compounds, as genes of selection of mycobacteria having received the shuttle vectors.
While one of the aims of this invention relates to the transfer of heterologous DNA to Mycobacterium, it should be noted that the shuttle vectors may also be used for transferring DNA fragments of mycobacteria to other bacteria such as Escherichia coli in order to facilitate the genetic study of the mycobacteria. It is a fact that mycobacteria, largely due to their slow growth rate, are difficult to study from a genetic point of view, and this study is facilitated by the cloning of fragments of their DNA in Escherichia coli. The shuttle vectors concerned by this invention may also be advantageously used for cloning the DNA of mycobacteria and for the study of these bacteria.
They may also be used to attenuate the virulence of M. tuberculosis and other virulent mycobacteria with the assistance of transposons.
This invention should thus considerably hasten progress in the genetic analysis of mycobacteria and will permit the genetic manipulation of BCG in order to fabricate multivalent vaccines.
For the purposes of this invention, xe2x80x9cheterologous DNAxe2x80x9d is understood to mean DNA not belonging to the species of Mycobacterium chosen to express it. As examples of transferable heterologous DNA, those described in EP 91.401.601 will be cited.
The genes of resistance to mercury and other mercurial compounds such as organo-mercurial compounds, are organized in operons.
Six genes mer have been described up to now for the transportation of Hg2+ ions in the cytoplasm and their detoxification.
The key enzyme is a mercuric reductase coded for the merA gene reducing the Hg2+ ions to Hg0 elemental mercury, a volatile compound of relatively little toxicity easily eliminated by the bacteria.
The gene merB is also known, a gene coding for an organo-mercurial lyase capable of splitting the C-Hg bond and of transforming the mercury in the Hg2+ ion which is then detoxified by the mercuric reductase described above.
All the genes of resistance to mercury which may be used within the scope of this invention, together with their sequences, may be those listed in reviews by Misra (1992, cited above), by Robinson and Tuoniven (1984, cited above), Fellay et al. (1987, Gene, 52, 147-154), Griffin et al. (1987, Proc. Natl. Acad. Sci. USA, 84, 3112-3116) or Herrero et al. (1990, J. Bacteriol., 172, 6557-6567).
Any other gene of resistance to mercury expressing itself in the mycobacteria may, however, be integrated in the shuttle vectors concerned by this invention.
The vector concerned by this invention advantageously comprises either the gene merA on its own or the genes merA and merB, with the genes merT and merP.
In the former case, the vector advantageously comprises the merA operon of the transposon Tn501. In the latter case, the vector advantageously comprises the operons merA and merB cloned from the plasmid pDU1358 isolated from Serratia marcescens (Griffin et al., cited above) and expressing a wide spectrum of resistance to the Hg2+ ion and to an organomercurial compound such as phenyl mercuric acetate (PMA).
Advantageously, a shuttle vector according to the invention and carrying the operon merA is the plasmid pMR001, filed with the Belgian Collections of Coordinated Microorganisms (BCCM) on May 18, 1994, under Accession No. LMBP 3046, and a vector carrying the two operons merA and merB is the plasmid pVN2 filed with the BCCM on Sep. 14, 1994, under Accession No. LMBP 3047. These two plasmids also carry the genes merT and merP, as well as merR for the pMR001.
Although genes of resistance to mercury are advantageously used in this invention, it should nonetheless be noted that any other gene of resistance to a heavy metal such as arsenic, cadmium or lead may also be used.
Genes giving resistance to these other heavy metals are notably those described by Silver and Walderhaug (Microbiological Reviews, Vol. 56, No. 1, 195-228, 1992), Kaur and Rosen (Plasmid 27, 29-40, 1992) and Nies (Plasmid, 27, 17-28, 1992).
The gene ars may be used in order to provide resistance to arsenic. A vector carrying such a gene is plasmid pVN3, filed with the BCCM on Sep. 14, 1994, under Accession No. LMBP 3048.
The gene(s) of resistance to the compound containing a heavy metal, as well as the enzyme cutting site, are preferentially carried by a transposon. Such a construction facilitates the integration of DNA carrying the cutting site in which has been inserted the DNA coding for the protein which it is desired to see expressed in the mycobacterium, particularly in cases when the homologous recombination is not very efficient.
As indicated above, said vector according to the invention comprises an enzyme cutting site allowing the insertion of DNA coding for a protein, capable of being expressed in the mycobacteria.
This invention thus relates not only to a vector having this cutting site but also to a vector in which a fragment of DNA coding for a protein heterologous to the chosen host has been inserted in this cutting site.
Such a protein is advantageously an antigen capable of inducing an immune response in the human or in the animal, such as an antigen of M. leprae, an antigen of M. tuberculosis, an antigen of the malaria vector, diphtheria toxoid, tetanus toxoid, an antigen of Leishmania, an antigen of a salmonella, an antigen of M. africanum, of M. intracellulae, of M. avium, an antigen of Treponema, of Toxoplasma, Schistosoma, Trypanosoma of Pertussis, an antigen of the herpes virus of HBV, of HCV, of Shigella, of Neisseria, of Borrelia, of the poliomyelitis virus, of the HIV virus, or an antigen of a venom of a snake, an insect or of Vibrio cholerae or any other antigen, including antigens coming from eukariotic cells, useful in immunoprophylaxis or immunotherapy.
The replication origins of the shuttle vector concerned by this invention are any origins allowing the replication of a vector in E. coli or in the mycobacteria. Advantageously, the replication origins in E. coli and in the mycobacteria are those used in the plasmid pRR3.
The origins of other plasmids such as pMSC262 and RSF1010 may also be advantageously used.
It should, however, be noted that if the origin is not functional in the mycobacteria, or if it is not optimally functional, the shuttle vector may integrate mycobacteria in the chromosomal DNA, for example by transposition or recombination.
The cutting site allowing the integration in the shuttle vector of the fragment of DNA coding for the protein which it is desired to express in the mycobacteria is any cutting site by a restriction enzyme making it possible to obtain sticky ends or blunt ends. Such an enzyme will be selected by those skilled in the art in the light of manipulation conditions such as ease of use of the enzyme.
Generally speaking, and particularly with reference to the production of the vector concerned by this invention and the integration of the DNA in this vector, those skilled in the art may refer to the following general technical manual: Maniatis et al. 1982, Molecular cloning: A Laboratory Manual; Cold Spring Harbor Laboratory, Cold Spring Harbor N.Y. USA or one of its recent re-editions.
Those skilled in the art may also refer to this manual for any matter concerning this invention.
This invention also relates to pathogenic mycobacteria, particularly M. bovis, M. leprae, M. tuberculosis, M. avium, M. intracellulare having a resistance to a compound containing a heavy metal such as inorganic mercury, a mercurial compound or arsenic.
In general, this invention relates to mycobacteria, in particular pathogenic or attenuated, carrying a vector such as described above.
It also relates to mycobacteria carrying a gene of resistance to a heavy metal and expressing a gene coding for a heterologous antigen.
xe2x80x9cheterologous antigenxe2x80x9d is understood as an antigen not usually found in the bacterium in which it is expressed.
The vector may be in the bacterium in a non-integrated form. However, it will be advantageously at least partly integrated in the bacterial chromosome.
It is especially advantageous for the mycobacteria to carry a transposon, containing the gene of resistance to the heavy metal and the gene coding for a protein capable of being expressed in the mycobacteria, inserted in their chromosome. Such a mode of application is particularly advantageous because it reduces the risks of loss of expression of the protein which may occur more frequently when the vectors are not integrated.
This invention also relates to pharmaceutical compositions containing at least one mycobacterium such as defined above in association with one or more compatible and pharmaceutically acceptable excipients and vaccines containing such mycobacteria.
Finally, this invention relates to a method of introducing a gene in a mycobacterium comprising at least one introduction step, in said mycobacterium, of a vector such as described above carrying said gene, and a step of selecting the mycobacteria in which the vector as introduced. The mycobacteria in which the plasmid has been introduced are advantageously selected for their resistance to mercury or a mercurial compound.
The vector is advantageously introduced in the mycobacteria by electroporation. However, any other method giving the same result may be used with equal success.